The long-range objective of our study is to define clearly the properties of mitochondrially bound forms of hexokinase in tumor cells and to establish both the roles of these enzymatic forms in the regulation of glucose utilization by neoplastic tissues and their requirement for tumor cell growth. We have established that the binding of hexokinase to mitochondria is a property of rapidly growing cancer cells. It is not a property of normal tissues like kidney, liver, and brain, where the small amount of hexokinase bound is associated with the endoplasmic reticulum. Future experiments will focus on six specific aims: (1)\to solubilize all the hexokinase activity associated with tumor mitochondria and to identify the total number of enzymatic species; (2)\to carry out submitochondrial localization studies to establish whether different enzymatic species have similar or different localizations; (3)\to purify mitochondrial forms of the enzymes from tumors characterized and tested for their ability to rebind to tumor and normal mitochondria; (4)\to modify enzymatically and chemically purified forms of mitochondrial hexokinase prior to rebinding in order to identify specific amino acid residues involved in binding; (5)\to solubilize and to attempt to purify and characterize mitochondrial receptors for hexokinase in tumor mitochondria; and (6)\to test Lonidamine, which inhibits the mitochondrial form of hexokinase in Ehrlich ascites cells, both for its ability to inhibit the mitochondrial form of the enzyme in other tumors and for its ability to suppress tumor cell growth in tissue culture and in tumor-bearing animals. (E)